Dopamine (DA) is a major neurotransmitter with diverse physiological impact, and is required for movement, sleep, working memory, and reward. Following evoked release, DA extracellular half-life is determined by presynaptic reuptake, mediated by the SLC6 plasma membrane DA transporter (DAT). Addictive and therapeutic psychostimulants, such as amphetamine, cocaine and methylphenidate (Ritalin), potently inhibit DA uptake, sustain DA signaling and impact DA-dependent behaviors. DAT coding variants are implicated in a variety of neuropsychiatric disorders, and transgenic mouse studies clearly demonstrate that DAergic signaling and behaviors, as well as psychostimulant efficacy, are highly sensitive to the level of DAT expression. DAT is not static at the plasma membrane, but is subject to robust constitutive and regulated endocytic recycling. However, it remains unclear whether regulated DAT internalization impacts DAergic function and/or DA-dependent behaviors. Our central hypothesis is that regulated DAT trafficking is likely a critical and influential determinant of DA signaling and DA-dependent behaviors. To test this hypothesis directly, we aim to replace endogenous mouse DAT with trafficking dysregulated DAT mutants in adult mice. However, germline DAT perturbations have clear developmental compensatory issues, underlining the need for a system that evaluates DAT mutants in vivo, but circumvents the pitfalls of germline mutant expression. To this end, we have developed a mouse model that integrates mouse with a floxed DAT gene (DATfl/fl) with a DAergic TET-OFF mouse (Pitx3IRES2-tTA). The resulting DATfl/fl;Pitx3IRES2-tTA mouse will facilitate AAV-mediated mouse DAT excision with Cre recombinase, and transgene replacement, driven by the Tet-responsive element, for in vivo molecular replacement studies. We contend that this system offers considerable advantages over germline knock-in approaches to test mutant protein function, as it circumvents developmental compensation and additionally allows for circuit-specific replacement using AAVs. Feasibility for this approach is strongly supported by strong preliminary data. The main aims of this two-year project are: 1) to optimize conditions for in vivo DAT molecular replacement, and 2) to test whether DAT ?gain-of-function? and ?loss of function? endocytic mutants impact DAT trafficking, DAergic signaling, and DA-dependent behaviors in male and female mice. We anticipate that at the completion of these studies we will have developed a powerful mouse system to interrogate the impact of DAT mutants in vivo in a manner that, heretofore, was not feasible. Moreover, we expect that our new mouse model will have broad utility for a number of researchers aiming to evaluate mutant function in adult mouse DAergic circuits.